Tuberculosis is a common and deadly infectious disease that is caused by mycobacteria, primarily Mycobacterium tuberculosis. Tuberculosis most commonly affects the lungs but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, bones, joints and even the skin. Other mycobacteria such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti can also cause tuberculosis, but these species do not usually infect healthy adults. Raviglione, et al. (2005). Harrison's Principle's of Internal Medicine, 953.
Over one-third of the world's population now has the tuberculosis bacterium in their bodies and new infections are occurring at a rate of one per second. World Health Organization (WHO). Tuberculosis Fact sheet No. 104—Global and regional incidence. March 2006, Not everyone who is infected develops the disease and asymptomatic latent tuberculosis infection is most common. However, one in ten latent infections will progress to active tuberculosis disease which, if left untreated, kills more than half of its victims. In 2004, 14.6 million people had active tuberculosis and there were 8.9 million new cases and 1.7 million deaths, mostly in developing countries. Drug-resistant strains of tuberculosis have emerged and are spreading (in 2000-2004, 20% of cases were resistant to standard treatments and 2% were also resistant to second-line drugs). “Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs—worldwide, 2000-2004”. MMWR Morb Mortal Wkly Rep 55 (11): 301-5.
In the patients where tuberculosis becomes an active disease, 75% of these cases affect the lungs, where the disease is called pulmonary tuberculosis. Symptoms include a productive, prolonged cough of more than three weeks duration, chest pain and coughing up blood. Systemic symptoms include fever, chills, night sweats, appetite loss, weight loss and paling, and those afflicted are often easily fatigued. When the infection spreads out of the lungs, extrapulmonary sites include the pleura, central nervous system in meningitis, lymphatic system in scrofula of the neck, genitourinary system in urogenital tuberculosis, and bones and joints in Pott's disease of the spine. An especially serious form is disseminated or miliary tuberculosis. Extrapulmonary forms are more common in immunosuppressed persons and in young children. Infectious pulmonary tuberculosis may co-exist with extrapulmonary tuberculosis, which is not contagious. Centers for Disease Control and Prevention (CDC), Division of Tuberculosis Elimination. Core Curriculum on Tuberculosis: What the Clinician Should Know. 4th edition (2000). Updated August 2003.
The primary cause of tuberculosis, Mycobacterium tuberculosis (MTB), is a slow-growing aerobic bacterium. Microbiology 150 (Pt 5): 1413-26. MTB is identified microscopically by its staining characteristics: it retains certain stains after being treated with acidic solution, and is thus classified as an “acid-fast bacillus” or AFB. Madison (2001) Biotech Histochem 76 (3): 119-25. In the most common staining technique, the Ziehl-Neelsen stain, AFB are stained a bright red which stands out clearly against a blue background. Acid-fast bacilli can also be visualized by fluorescent microscopy, and by an auramine-rhodamine stain.
Tuberculosis is spread by aerosol droplets expelled by people with the active disease of the lungs. Transmission can only occur from people with active—not latent—TB disease. The probability of transmission from one person to another depends upon the quantity of the infectious droplets expelled by the patient, the effectiveness of ventilation, the duration of exposure, and the virulence of the Mycobacterium tuberculosis strain. Centers for Disease Control and Prevention (CDC), Division of Tuberculosis Elimination. Core Curriculum on Tuberculosis: What the Clinician Should Know. 4th edition (2000). The chain of transmission can therefore be broken by isolating patients with active disease and starting effective anti-tuberculous therapy.
Tuberculosis is classified as one of the granulomatous inflammatory conditions. Macrophages, T lymphocytes, B lymphocytes and fibroblasts are among the cells that aggregate to form a granuloma, with lymphocytes surrounding the infected macrophages. The granuloma functions not only to prevent dissemination of the mycobacteria, but also provides a local environment for communication of cells of the immune system. Within the granuloma, T lymphocytes (CD4+) secrete cytokines such as interferon gamma, which activates macrophages to destroy the bacteria with which they are infected. Kaufmann (2002) Ann Rheum Dis 61 Suppl 2: ii54-8. T lymphocytes (CD8+) can also directly kill infected cells. Houben et al. (2006) Curr Opin Microbiol 9 (1): 76-85.
HIV-1-infection markedly reduces host defense and increases disease progression in the absence of DOTS, highlighting the role of cellular immunity in controlling mycobacteria. Transgenic knockout models of mycobacteria-exposed mice demonstrated that interferon-γ and its signaling intermediates are critical to host defense (Flynn et al., J Exp Med 1993; 178:2249-2254; Kamijo et al., J Exp Med 1993; 178:1435-1440). In humans, mutational defects in the interferon-γ receptor, the cytokine IL-12, or antibodies to interferon-γ result in disseminated mycobacterial infection (Seneviratne et al., Thorax 2007; 62:97-99; Dorman et al., Lancet 2004; 364:2113-2121). Treatment of these patients with recombinant interferon-γ has been successful and safe.
Responses of whole blood PBMC to PPD and other mycobacterial antigens are reduced in active tuberculosis probably due to suppressor T cells (Tregs or Th17 cells) or cytokines, e.g. IL-10 or TGF-β (Hirsch et al., J Infect Dis 1999; 180: 2069-73; Hougardy et al., Am J Respir Crit Care Med 2007; 176: 409-416). Bronchoalveolar lavage (BAL) of patients with pulmonary tuberculosis has shown increases in interferon-γ and percent CD4+ cells compared to uninfected controls, but in advanced, cavitary tuberculosis or TB/HIV-1 coinfection these markers of effective immunity are reduced (Law et al., Am J Respir Crit Care Med 1996; 153:1377-1384; Condos et al., Am J Respir Crit Care Med 1998; 157:729-735).
Progression from tuberculosis infection to tuberculosis disease occurs when the tuberculosis bacilli overcome the immune system defenses and begin to multiply. In primary tuberculosis disease—1 to 5% of cases—this occurs soon after infection. However, in the majority of cases, a latent infection occurs that has no obvious symptoms. These dormant bacilli can produce tuberculosis in 2 to 23% of these latent cases, often many years after infection. Parrish et al., (1998) Trends Microbiol 6 (3): 107-12. The risk of reactivation increases with immunosuppression, such as that caused by infection with HIV. In patients co-infected with M. tuberculosis and HIV, the risk of reactivation increases to 10% per year. Onyebujoh et al. World Health Organization Disease Watch: Focus: Tuberculosis. December 2004.
Treatment for tuberculosis uses antibiotics to kill the bacteria. The two antibiotics most commonly used are rifampicin or rifampin and isoniazid. However, these treatments are more difficult than the short courses of antibiotics used to cure most bacterial infections as long periods of treatment (around 6 to 12 months) are needed to entirely eliminate mycobacteria from the body. Centers for Disease Control and Prevention (CDC), Division of Tuberculosis Elimination. Core Curriculum on Tuberculosis: What the Clinician Should Know. 4th edition (2000). Latent tuberculosis treatment usually uses a single antibiotic, while active tuberculosis disease is best treated with combinations of several antibiotics, to reduce the risk of the bacteria developing antibiotic resistance. O'Brien (1994) Semin Respir Infect 9 (2): 104-12. People with these latent infections are treated to prevent them from progressing to active tuberculosis disease later in life. However, treatment using Rifampin and Pyrazinamide is not risk-free. The Centers for Disease Control and Prevention (CDC) notified healthcare professionals of revised recommendations against the use of rifampin plus pyrazinamide for treatment of latent tuberculosis infection, due to high rates of hospitalization and death from liver injury associated with the combined use of these drugs. (2003) MMWR Morb Mortal Wkly Rep 52 (31): 735-9.
Drug resistant tuberculosis is transmitted in the same way as regular tuberculosis. Primary resistance occurs in persons who are infected with a resistant strain of tuberculosis. A patient with fully-susceptible tuberculosis develops secondary resistance (acquired resistance) during tuberculosis therapy because of inadequate treatment, not taking the prescribed regimen appropriately, or using low quality medication. O′Brien (1994) Semin Respir Infect 9 (2): 104-12. Drug-resistant tuberculosis is a public health issue in many developing countries, as treatment is longer and requires more expensive drugs. Multi-drug resistant tuberculosis (MDR—tuberculosis) is defined as resistance to the two most effective first line tuberculosis drugs: rifampicin and isoniazid. Extensively drug-resistant tuberculosis (XDR-TB) is also resistant to three or more of the six classes of second-line drugs. (2006) MMWR Morb Mortal Wkly Rep 55 (11): 301-5.
Clinical trials of recombinant interferon-γ (rIFN-γ) in humans are few. As of 1999, IFN-γ is indicated for the treatment of chronic granulomatous disease in which prolonged treatment (average duration 2.5 years) was associated with improvement in skin lesions, with minimal adverse events (fever, diarrhea, and flu-like illness) (N Engl J Med 324 (8):509-16; Bemiller et al. (1995) Blood Cells Mol Dis 21(3): 239-47; Weening et al., (1995) Eur J Pediatr 154(4):295-8). Boguniewicz treated 5 patients with mild atopic asthma with escalating doses of aerosolized r IFN-γ (maximum dose of 500 mcg, total study dose of 2400 mcg) delivered over 20 days (Boguniewicz et al., (1995) J Allergy Clin Immunol 95(1) Pt 1: 133-5). All patients tolerated the nebulized r IFN-γ but there were no significant changes in the endpoints evaluated which included peak flow. InterMune sponsored several clinical trials to evaluate IFN-γ for infectious diseases. The MDR-TB clinical trial was entitled “A Phase II/III Study of the Safety and Efficacy of Inhaled Aerosolized Recombinant Interferon-γ 1 b in Patients with Pulmonary Multiple Drug Resistant Tuberculosis (MDR-TB) Who have Failed an Appropriate Three Month Treatment.” This study enrolled 80 MDR-TB patients at several sites and randomized them to receive aerosol rIFN-γ (500 μg MWF) or placebo for at least 6 months in addition to second line therapy. This clinical trial was stopped prematurely due to lack of efficacy on sputum smears, M tb culture, or chest radiograph changes.
We administered nebulized rIFN-γ to 5 patients with persistent acid fast bacilli (AFB) smear and culture positive multiple-drug resistant tuberculosis (TB) (Condos et al., (1997) Lancet 349(9064): 1513-5). Patients received aerosol r IFN-γ, 500 mcg, 3 times weekly for 4 weeks (total study dose 6000 mcg). Therapy was tolerated well with minimal side effects. At the end of the 4 weeks, 4 of the 5 patients were sputum AFB-smear negative and the time to positive culture increased indicating a reduced organism load after treatment. Interestingly, in these reported and in additional patients, PEFR performed 1 hour after treatment improved by 6% (n=10).
Sputum culture positive at two months, cavitation on chest radiography, being underweight, and bilateral pulmonary involvement increases the risk of treatment failure and/or relapse (Tuberculosis Trials Consortium (2002) Lancet 360: 528-534. Responses of whole blood PBMC to PPD and other mycobacterial antigens are reduced in active tuberculosis probably due to suppressor T cells (Tregs) or cytokines, e.g. IL-10 or TGF-β (Hirsch, et al. (1999) J Infect Dis 180: 2069-2073; Hougardy, et al. (2007) Am J Respir Crit Care Med 176: 409-416.) Bronchoalveolar lavage (BAL) of patients with pulmonary tuberculosis has shown increases in inflammatory cytokines and percent CD4+ cells compared to uninfected controls, and in advanced, cavitary tuberculosis markers of effective immunity are reduced. Nitric oxide (NO) has been identified as a mechanism of mycobacterial killing, while mycobacteria can also be eliminated by autophagy, apoptosis, cytotoxic CD8+ cells, alpha defensins from neutrophils, and phagosomal-lysosomal rupture with subsequent cytosolic demise (Martineau, et al. (2007) J Clin Invest 117: 1988-1994; Van der Wel, et al. (2007) Cell 129: 1287-1298; Gutierez, et al. (2004) Cell 119: 753-766). However, mycobacteria have evolved virulence factors to persist in macrophages, possibly disrupting the interferon-γ signaling pathways.
Interferons are a family of naturally-occurring proteins that are produced by cells of the immune system. Three classes of interferons have been identified, alpha, beta and gamma. Each class has different effects though their activities overlap. Together, the interferons direct the immune system's attack on viruses, bacteria, tumors and other foreign substances that may invade the body. Once interferons have detected and attacked a foreign substance, they alter it by slowing, blocking, or changing its growth or function.
Interferon-γ is a pleiotropic cytokine that has specific immune-modulating effects, e.g. activation of macrophages, enhanced release of oxygen radicals, microbial killing, enhanced expression of MHC Class II molecules, anti-viral effects, induction of the inducible nitric oxide synthase gene and release of NO, chemotactic factors to recruit and activate immune effector cells, downregulation of transferrin receptors limiting microbial access to iron necessary for survival of intracellular pathogens, etc. Genetically engineered mice that lack interferon-γ or its receptor are extremely susceptible to mycobacterial infection.
Recombinant IFN-γ was administered to normal volunteers and cancer patients in the 1980s through intramuscular and subcutaneous routes. There was evidence of monocyte activation, e.g. release of oxidants. Jaffe et al. reported rIFNγ administration to 20 normal volunteers. (See, Jaffe et al., J Clin Invest. 88, 297-302 (1991)) First, they gave rIFN-γ 250 μg subcutaneously noting peak serum levels at 4 hours and a trough at 24 hours.
Several clinical trials were sponsored to evaluate IFN-γ for infectious diseases. The MDR-TB clinical trial, entitled “A Phase II/III Study of the Safety and Efficacy of Inhaled Aerosolized Recombinant Interferon-γ 1 b in Patients with Pulmonary Multiple Drug Resistant Tuberculosis (MDR-TB) Who have Failed an Appropriate Three Month Treatment,” enrolled 80 MDR-TB patients at several sites (Cape Town, Port Elizabeth, Durban, Mexico) and randomized them to receive aerosol rIFN-γ (500 μg MWF) or placebo for at least 6 months in addition to second line therapy. This clinical trial was stopped prematurely due to lack of efficacy on sputum smears, Mtb culture, or chest radiograph changes.
Ziesche et al. gave rIFN-γ subcutaneously at a dose of 200 mg three times a week in addition to oral prednisone to 9/18 patients with idiopathic pulmonary fibrosis (IPF). See, Ziesche et al., (1999) N. Eng. J. Med., 341, 1264-1269). The results of a subsequent phase 3 clinical trial of interferon γ-1b therapy for IPF were recently published. Although this was the first clinical trial of IPF that had an adequate sample size and was a randomized, prospective, double-blind, placebo-controlled study, no significant effect on markers of physiologic function, such as forced vital capacity, was observed. However, more deaths occurred in the placebo group, and survival was significantly better for a subset of patients who received interferon γ-1b therapy and had a forced vital capacity of 55% or greater and diffuse lung capacity for carbon monoxide of 35% or greater of the normal predicted values. The discordance between disease progression and survival in that study remains to be explained. One possibility is that interferon γ-1b therapy improves host defense against infection and diminishes the severity of lower respiratory tract infection when it complicates the clinical course of patients with IPF. This possibility is supported by the observation by Strieter et al. that the interferon-inducible CXC chemokine, I-TAC/CXCL11, which has antimicrobial properties, was significantly up-regulated in plasma and bronchoalveolar lavage (BAL) fluid in individuals who received interferon γ-1b compared to those who received placebo, whereas profibrogenic cytokines were generally not significantly altered by interferon γ-1b therapy over a 6-month treatment period. (See, Strieter et al., Am J Respir Crit Care Med. (2004). One possibility to explain the lackluster results is inadequate levels of drug delivered to the lung interstitium with current dosing strategies.
Interferon-γ is as essential for human host response to Mtb as it is in knockout mouse models. In a report of a patient with severe interferon-γ deficiency who developed disseminated TB, complete healing of skin, bone, pleural and renal lesions was achieved with subcutaneous IFN-γ1b three times a week at 50 μg for 34 weeks and 75 μg for 16 weeks (Seneviratne et al., Thorax (2007) 62:97-99). In another patient with autoimmune type I diabetes, primary hypothyroidism, and fatal disseminated Mtb infection, autoantibodies to interferon-γ that were capable of blocking in vitro responses to IFN-γ by peripheral blood mononuclear cells from normal donors were detected (Doffinger et al., Clin Inf Dis (2004) 38:e10-e14). Treatment of disseminated nontuberculous mycobacterial infection with subcutaneous IFN-γ has been clinically successful with correction of in vitro antigen-stimulated PBL responses (Holland et al., N Engl J Med (1994) 330: 1348-55). A patient with disseminated skin lesions due to M abscessus after chemotherapy for essential thrombocytosis had complete clearing of the skin after 7 months of subcutaneous IFN-γ, and a mid-course biopsy showed prominent MHC Class II immunostaining (Colsky et al., Arch Dermatol (1999) 135:125-127).
An open-label study of nebulized IFN-γ1b in 5 patients with MDR-TB showing clearing of the sputum after 1 month, as well as a second study in 10 TB patients showing enhanced IFN-γ signaling after one month of therapy with nebulized IFN-γ1b was previously reported (Condos et al., Lancet (1997) 349:1513-1515; Condos et al., Infect Immun (2003) 71: 2058-2064.). The IFN-γ inducible IP-10 was increased in BAL following nebulized IFN-γ1b but that nitric oxide synthase 1 (NOS2) was not increased further since over two-thirds of alveolar macrophages expressed this in active pulmonary TB at onset of treatment (Raju et al., Infect Immun (2004) 72: 1275-1283; Nicholson et al., J Exp Med (1996) 183: 2293-2302). Similar results were reported for 8 MDR-TB patients treated with intramuscular IFN-γ1b over a 6-month period (Suárez-Méndez et al., BMC Infect Dis (2004) 4:44). In a further study, nebulized interferon-α was given for 2 months as an adjunct in a randomized controlled clinical trial in 20 tuberculosis patients (Giosuè et al., Am J Respir Grit Care Med (1998) 158:1156-1162). Improvements in fever, M tuberculosis number in the sputum, abnormalities in CT-scans, and more significant decreases in BALF IL-1β, IL-6, and TNF-α were noted in the IFN-α group (Palmero et al., Int J Tuberc Lung Dis (1999) 3: 214-218). In a clinical trial of interleukin-2 adjunctive therapy performed in Uganda, 110 TB patients were randomized to IL-2 subcutaneously twice/day for the first 30 days in addition to DOTS versus DOTS alone. Seventeen percent versus 30% sputum conversion, respectively, was observed at 4 weeks (Johnson et al., Am J Respir Crit Care Med (2003) 168: 185-191). IL-2 might have upregulated CD4+CD25+ T regulatory cells, which could have down-regulated the immune system and impaired clearance of organisms.
The inflammatory response in the lung in TB may include an increase in neutrophils, particularly in radiographically abnormal areas (Law et al., Am J Respir Crit Care Med (1996) 153: 799-804). These involved segments have BAL cell supernatants and fluid that contain increased levels of IL-1β, IL-6, TNF-α, and IL-8, a chemokine for neutrophils (Law et al., Am J Respir Crit Care Med (1996) 153: 799-804; Zhang et al., J Clin Invest (1995) 95: 586-592). Mtb and its cell wall components lipoarabinomannan (LAM), lipomannan (LM), and phosphoinositolmannoside (PIM) stimulated IL-8 protein release and mRNA expression in vitro from alveolar macrophages (Zhang et al., J Clin Invest (1995) 95: 586-592). In a series of 30 pulmonary tuberculosis patients undergoing bronchoalveolar lavage, a sub-group had minimal disease with increased BAL lymphocytes with IFN-γ release, and a sub-group with cavitary tuberculosis had exaggerated percent and numbers of neutrophils with increased TNF-α and IL-1-β in BALF (Hougardy et al., Am J Respir Crit Care Med (2007) 176: 409-416). Neutrophils kill Mtb by oxidative and nonoxidative mechanisms probably by release of alpha defensins and bactericidal permeability increasing protein (Zhang et al., J Clin Invest (1995) 95: 586-592). Recently, human neutrophil peptides 1-3 were shown to kill Mtb and cathelicidin LL-37 and lipocalin 2 restricted growth of the organism, with the latter in an iron-dependent manner (Martineau et al., J Clin Invest (2007) 117: 1988-1994). Monocytes and macrophages are recruited to the site of Mtb inflammation by the release of the CC chemokines CCR2, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α and -1β (Schwander et al., J Infect Dis (1996) 173: 1267-1272). Other mechanisms of Mtb killing may be modulated by IFN-γ include autophagy and ubiquitin in lysosomes (Alonso et al., Proc Natl Acad Sci (2007) 104: 6031-6036). IFN-γ-inducible chemokines CCXCL-9,-10, and -11 were increased in lung granulomas of cynmologus macaques infected with Mtb, and TNF-α, IFN-γ, and CD3+ cells were also abundant (Fuller et al., Infect Immun (2003) 71:7023-7034). CXCL-11 was also increased in BAL fluid from patients with idiopathic pulmonary fibrosis treated with subcutaneous IFN-γ1b (Strieter et al., Am J Respir Crit Care Med (2004) 170: 133-140).
Mtb usually produces an environment of immunological depression or stasis with reduced peripheral blood lymphocyte response to Mtb antigens that improves after treatment and becomes polarized to a Th1 profile of cytokines (Lienhardt et al., Eur J Immunol (2002) 32: 1605-1613). Treg or CD4+CD25+ cells are expanded in active tuberculosis and decline after treatment with concomitant return of T cell responses to PPD and release of IFN-γ (Condos et al., Am J Respir Crit Care Med (1998) 157:729-735). BAL lymphocytes and macrophages have increased mRNA transcripts for IFN-γ and IL-12 at the onset of active pulmonary tuberculosis (Robinson et al., Am J Respir Crit Care Med (1994) 149: 989-993). IL-18 synergizes with IL-12 in inducing IFN-γ, and correlates with fever and extent of radiographic tuberculous disease (Yamada et al., Am J Respir Crit Care Med (2000) 161: 1786-1789).